Although P. carinii is a common opportunistic pathogen causing pneumonia in hundreds of thousands of immunocompromised patients, many basic questions about the organism and the disease it causes remain unanswered. Do different strains of P. carinii infect humans? Are patients with recurrent P. carinii infections suffering relapse or reinfection? Do strains resistant to antimicrobials exist and if so, what mechanisms of resistance occur? Answers to these questions may greatly influence clinical practice. Using modern tools of molecular biology and immunology and the greatly expanded patient pool resulting from the AIDS epidemic, these questions can now be addressed. The first aim of this proposal is to determine the degree of variation in the genome of P. carinii from patient specimens acquired in ACTUs at Indiana University and other ACTU sites. Sequences obtained from PCR (polymerase chain reaction) of up to 4 regions of the genome from 30 selected P. carinii isolates will be tested for differences related to geographical origin of patients. P. carinii in the 30 selected BAL (bronchoalveolar lavage) specimens will also be tested with monoclonal and polyclonal antibodies that discriminate among human isolates. The second aim is to compare isolates from patients with recurrent infection to isolates from their initial infection to determine if the recurrence is caused by relapse due to the same strain or reinfection with a different strain. Up to two hundred BAL specimens taken from patients suffering initial infections will be saved each year under conditions suitable to allow later use for PCR; these specimens serve as controls against which to compare isolates taken from later infections. Under the third aim P. carinii from specimens acquired in ACTUs at Indiana University and elsewhere will be compared for molecular differences that may alter susceptibility to trimethoprim/sulfamethoxazole. Portions of rat P. carinii dihydrofolate reductase gene will be used as a probe for PCR, to augment and isolate the dihydrofolate reductase gene from P. carinii in human BAL samples. Using RFLP (restriction fragment length polymorphism) and partial sequencing, homology of isolates from untreated patients and from patients failing therapy will be assessed. Any dihydrofolate reductase genes from human P. carinii showing different sequences will be expressed in dhfr E. coli and the functional consequences of these differences will be determined. The results of the experiments described should allow determination of 1) the inherent degree of genetic diversity in human P. carinii isolates from several geographical areas, 2) the possibility of reinfection versus relapse, 3) the molecular differences in P. carinii from patients failing therapy with trimethoprim/sulfamethoxazole.